Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Recent findings suggest that HDACs could be key targets for chemotherapeutic intervention in malignant diseases. A convenient and sensitive fluorogenic assay for HDAC activity would therefore expedite studies of HDAC in transcriptional regulation and in vitro screening for drug discovery. In this study, novel fluorogenic substrates of HDACs were synthesized with an epsilon-acetylated lysyl moiety and an adjacent MCA moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules became substrates for trypsin, which released highly fluorescent AMC molecules in a subsequent step of the assay. The fluorescence increased in direct proportion to the amount of deacetylated substrate molecules, i.e., HDAC activity. The nonisotopic, homogeneous assay is well suited for high-throughput HDAC inhibitor screening.